Process for the preparation of antibiotic W-10 complex and for the isolation of Antibiotic 20561 and Antibiotic 20562 therefrom

ABSTRACT

A novel antibiotic complex exhibiting antifungal activity is produced by fermenting a novel species of bacteria from the genus Aeromonas, the bacterium being herein designated Aeromonas sp. W-10. The antibiotic complex is comprised of at least three and possibly as many as six components and has been designated Antibiotoc W-10 Complex. Two members of the complex have been isolated in substantially pure form and are designated Antibiotic 20561 and Antibiotic 20562, the antibiotics exhibiting substantial antifungal activity.

This is a division of application Ser. No. 767,102, filed Feb. 9, 1977 now U.S. Pat. No. 4,137,224.

This invention relates to a novel bacterium and to an antifungal mixture produced thereby. More particularly, this invention relates to a bacterium of the genus Aeromonas, herein designated Aeromonas sp. W-10 and to the novel antibiotic mixture produced thereby.

THE MIXTURE

Cultivation of Aeromonas sp. W-10 under controlled aerobic conditions is a aqueous nutrient medium produces a mixture having antifungal activity. The mixture is herein designated "Antibiotic W-10 Complex". The complex consists of from three to about six discreet entities, two of which have been isolated free from the compounds coproduced therewith. These compounds exhibit substantial antifungal activity and are designated, "Antibiotic 20561" and "Antibiotic 20562".

Antibiotic 20561 is a solid composition of matter having the following physical properties:

(a) A melting point of 160°-166° C.;

(b) A specific optical rotation as measured at the D line of sodium at 26° C. of -88°±3° at 0.4% concentration in pyridine containing 5% water, and of -66°±2° in dimethylsulfoxide containing 5% water at 0.4% concentration;

(c) Ultraviolet absorption maxima in methanol at 220 and 280 mμ with E_(1cm) ^(1%) of 313 and 20, respeactively; and in methanol containing 10% v/v 1N sodium hydroxide, maxima are observed at 240 and 294 mμ;

(d) A nuclear magnetic spectrum is deuterated dimethylsulfoxide--as set forth below.

(e) An infrared absorption spectrum in mineral oil (Nujol) substantially as shown in Table IV;

(f) A molecular formula of C₅₇ H₈₆ O₁₀ N₁₂ ; and having the following structural formula: ##STR1##

Antibiotic 20562 is a solid composition of matter having the following physical properties:

(a) A melting point of 170°-175° C.;

(b) A specified optical rotation as measured at the D line of sodium at 26° C. of -60°±3° in pyridine containing 5% water at 0.4% concentration, and is -44°±3° in dimethylforamide containing 5% water at 0.4% concentration;

(c) Ultraviolet absorption maxima in methanol at 222 and 280 mμ with E_(1cm) ^(1%) of 268 and 14.8, respectively, and in methanol containing 10% v/v 1N sodium hydroxide, at 240 and 292 mμ with E_(1cm) ^(1%) of 190 and 19.4, respectively;

(d) An infrared absorption spectrum in mineral oil (Nujol) substantially as shown in FIG. 1;

(e) A nuclear magnetic resonance spectrum is deuterated dimethylsulfoxide substantially as shown in FIG. 2;

(f) A molecular formula of C₆₃ H₉₆ O₂₁ N₁₂ ; and having the following structural formula:

THE MICROORGANISM

Based upon the physiological characteristics, morphology and the guanine plus cytosine content of isolated DNA, we have classified the organism as a novel member of the genus Aeromonas. We have designated the bacterium Aeromonas sp. W-10, sometimes referred to herein as Aeromonas W-10, and have deposited the microorganism with the United States Department of Agriculture, Northern Utilization Research and Development Division (NRRL), Peoria, Ill., from whom subcultures are available. The microorganism has been assigned the numerical designation NRRL B-11053. Although this invention is described in terms of Aeromonas sp. W-10, it is not limited to the particular strain on deposit but embraces Antibiotic W-10 producing mutants and variants obtained by the use of mutagenic agents, and by the use of methods known to the art such as exposing the microorganism to X-ray or ultraviolt radiation, or to nitrogen mustards, or to bacteriophages of the like. When observed microscopically, Aeromonas sp. W-10 is seen to be a non-spore forming, gram-negative rod approximately 0.5 to 1.0×1.0-1.5 microns. It occurs singly and is motile by means of a single polar flagellum. The organism is faculatively anaerobic, oxidase positive, capable of reducing nitrates to nitrites, insensitive to the vibriostatic compound 2,4-diamino-6,7-diisopropylpteridine (0/129) with a guanine-cytosine content of DNA at 61%. According to Bergy's Manual of Determinative Bacteriology, 8th Edition, the organism is, on the basis of the data set forth, classified as a member of the genus Aeromonas.

The growth and physiological characteristics of Aeromonas sp. W-10 on various media are presented in the following tables. All observations were made after seven (7) days of incubation at 28° C. Color designations are assigned according to the classification in the Color Harmony Manual (1958) , Container Corporation of America. The designates G, S, C and R used herein for growth, surface, color and reaction, respectively.

                  TABLE I                                                          ______________________________________                                         Growth Characteristics of Aermonoas sp. W-10 on Various Media                  Medium            Description                                                  ______________________________________                                         Bennett's Agar    G: +++, good                                                                   S: flat, no aerial mycelium; no                                                  diffusable pigment                                                           C: g4pg, dark luggage tan                                    Calcium Citrate Agar                                                                             G: +, fair                                                                     S: plicate, no aerial mycelium;                                                  no diffusable pigment                                                        C: g3ic, light amber                                         Calcium Malate Agar                                                                              G: +, fair                                                                     S: plicate, no aerial mycelium;                                                  no diffusable pigment                                                        C: g 11/2 Ca, cream                                          Czapek's Glucose Solution                                                                        G: ±, poor                                                Czapek's Sucrose Agar                                                                            G: ±, poor                                                Emerson's Agar    G: +++, good                                                                   S: flat to plicate, no aerial                                                    mycelium; no diffusable pig-                                                   ment                                                                         C: g3ec, light beige                                         Glucose-Asparagine Agar                                                                          G: ++, moderate                                                                S: flat, no aerial mycelium;                                                     no diffusable pigment                                                        C: g2eg, beige                                               Glucose-Yeast Extract                                                                            G: +++, good                                                 Agar              S: flat to plicate, no aerial                                                    mycelium; no diffusable pig-                                                   ment                                                                         C: center g3ec, light beige                                                      periphery-g4pe,                                                                orange rust                                                Nutrient Agar     G: +, fair                                                                     S: flat, no aerial mycelium;                                                     no diffusable pigment                                                        C: g2ne, luggage tan                                         NZ Amine Glucose Agar                                                                            G: +++, good                                                                   S: flat to plicate, no aerial                                                    mycelium                                                                     C: center-g4ng, saddle tan                                                       periphery-g3ec, light beige                                Ordinary Agar     G: ±, poor                                                Peptone Glucose Agar                                                                             G: +++, good                                                                   S: plicate, no aerial mycelium,                                                  no diffusable pigment                                                        C: g4ne, luggage tan                                         Potato Slice      G: +++, good                                                                   S: flat, no aerial mycelium; no                                                  diffusable pigment                                                           C: g3ie, camel                                               Starch Agar (Waksman No. 21)                                                                     G: ±, poor                                                Tomato Paste      G: +++, good                                                 Oatmeal Agar      S: flat to plicate, no aerial                                                    mycellium; no diffusable                                                       pigment                                                                      C: g4pg, dark luggage tan                                    ______________________________________                                    

                  TABLE II                                                         ______________________________________                                         Growth characteristics and physiological properties of                         Aeromonas W-10 on various media                                                Medium         Description                                                     ______________________________________                                         Egg Agar (Dorset's)                                                                           G: ++, moderate                                                                S: flat, no aerial mycelium;                                                     no diffusable pigment                                                        C: g2ca, ivory                                                                 R: no hydrolysis-negative                                       Loeffler's Serum Medium                                                                       G: +++, good                                                                   S: flat, no aerial mycelium;                                                     no diffusable pigment                                                        C: g3ie, camel                                                                 R: Serum partially liquified-                                                    positive                                                      Gelatin Agar (McDade)                                                                         G: +++, good                                                                   S: plicate, no aerial mycelium;                                                  no diffusable pigment                                                        C: g3ie, camel                                                                 R: Galatin hydrolyzed-positive                                  Starch Agar    G: +++, good                                                                   S: flat, no aerial mycelium,                                                     no diffusable pigment                                                        C: g3ec, light beige                                                           R: Starch weakly hydrolyzed                                     Tyrosine Yeast Agar                                                                           G: +++, good                                                                   S: plicate, no aerial mycelium,                                                  no diffusable pigment                                                        C: g3pi, tobacco brown                                                         R: Tyrosine crystals dissolved-                                                  positive                                                      Tyrosine Beef Agar                                                                            G: +, fair                                                                     S: flat, no aerial mycelium, no                                                  diffusable pigment                                                           C: g3cc, light beige                                                           R: Tyrosine crystals dissolved-                                                  positive                                                      ______________________________________                                    

                  TABLE III                                                        ______________________________________                                          Physiological Characteristics of Aeromonas sp. W-10                                       Re-                                                                Test        sult     Test         Result                                       ______________________________________                                         Acid Production.sup.1                                                          From:                                                                          Adonitol    -      Fermentation of:                                            D-Arabinose -      Glucose        +                                            L-Arabinose -                                                                  Cellibiose  -      Hydrolysis of:                                              Dextrin     +      Gelatin        +                                            Dulcitol    -      Casein         +                                            Erythritol  -      Starch         ± (weak)                                                     Egg            -                                            Fructose    +      Serum          +                                            Galactose   -      Cellulose      - → ±                              Glucose     +      Tyrosine       +                                            Glycerol    +      Reactions:                                                  Glycogen    +      Litmus Milk    Coagulation,                                 Inositol    +                     slight pepton-                                                                 ization                                      Inulin      +                                                                  Lactose     -      Nitrate to                                                  Maltose     -      Nitrite        positive                                     Mannitol    +                                                                  Mannose     -      Indole         -                                            Melibiose   -      Catalase       +                                            Raffinose   -      Oxidase        +                                            Rhamnose    -      Arginine                                                    Ribose      -      Hydrolase      +                                            Sorbitol    -      Lysine                                                      Sucrose     -      Decarboxylase  -                                            Trehalose   -      Ornithine                                                   D-xylose    -      Decarboxylase  -                                                               Urease         -                                                               Growth At:                                                                     pH 6           +                                                               28 C           +                                                               37 C           ± → +                                                 45 C           -                                                               Production of:                                                                 H.sub.2 S      -                                                               Melanin        -                                            Resistance to:                                                                 0/129 (300 mcg)                                                                            +      NaCl Tolerance:                                                                               2-3%                                         Polymyxin (50 mcg)                                                                         +                                                                  Penicillin G (10 u)                                                                        +      Diaminopimelic Acid                                         Pacitracin (10 u)                                                                          +      in whole cells + (meso)                                     Gentamicin (10 mcg)                                                                        -                                                                  Sisomiein (10 mcg)                                                                         -                                                                  Naladixic Acid                                                                             -      % Guanine-Cytosine                                                                            61% ± 2%                                  (50 mcg)                                                                       ______________________________________                                          .sup.1 Gas not formed in the aerobic breakdown of carbohydrates.         

THE FERMENTATION

Although Aeromonas sp. W-10 will grow on a wide variety of nutrients, the following are preferred for preparing the vegetative inoculum: trypticase, soy peptone, beef infusion, casamino acids, dextrose, soluble starch and inorganic salts such as sodium chloride and dipotassium phosphate.

Similarly, Aeromonas sp. W-10 will produce the Antibiotic W-10 Complex in a nutrient medium containing any of a wide variety of nutrients. However, the following are preferred: casamino acids, soluble starch, soybean meal, dextrose, buffers e.g. calcium carbonate and inorganic salts, such as cobalt chloride.

The production of Antibiotic W-10 Complex is commenced by the preparation of a vegetative inoculum of Aeromonas sp. W-10 in a suitable nutrient medium from an agar slant or of a portion of a frozen broth culture. The nutient medium is a conventional one containing assimilable sources of nitrogen and carbon and is usually inoculated with a 5% v/v transfer of frozen broth culture. The inoculum preparation is preferably effected at a temperature in the vicinity of 35° C. for from about 25 to about 48 hours with aeration and agitation. For production-sized fermentations, a second inoculum preparation is recommended.

The fermentation may be effected in a medium having the same constituents as the inoculum medium or in one containing different nutrients and is preferably effected at a temperature in the vicinity of 28° , a pH of about 6.5-7.0 using a 5% v/v inoculum. The fermentation is complete in from about 48 to about 90 hours, usually from about 48 to about 72 hours with aeration and agitation.

Peak Antibiotic W-10 Complex production is determined by periodic sampling of the fermentation mixture and by subjecting the samples to an agar diffusion assay using Saccharomyces cerevisiae ATCC 9763 as the test organism.

The broths are assayed against a standard solution of Antibiotic 20562 having an assigned potency of 1000 mcg/mg. The standard solution is diluted with 10% potassium phosphate buffer (pH 6.0) to make solutions having antibiotic concentrations of 3.2, 4.0, 5.0, 6.25 and 7.8 mcg/ml. The assay is performed substantially as set forth in the Federal Register, Title 21, Chapter 436.105.

THE ANTIBIOTICS

When peak Antibiotic W-10 Complex activity is attained, the fermentation mixture is extracted with a polar water immiscible organic solvent, preferably butanol saturated with water. The extracts are combined and concentrated, preferably in vacuo until a precipitate forms wherein the suspension is filtered. The filtrate is concentrated to a residue, redissolved in methanol and added to a 1:1 mixture of petroleum ether:ethyl ether with agitation. The resulting precipitate is removed by filtration and dried in vacuo.

The precipitate removed during the concentration of the butanol extracts and the residue obtained by concentration of the filtrate both contain Antibiotic 20561, Antibiotic 20562 in admixture with biologically inactive organic matter and the other unidentified active substances forming the Antibiotic W-10 Complex. Separation of Antibiotic 20561 and Antibiotic 20562 is best accomplished by chromatographic means after a suitable pre-treatment to remove grossly different materials.

The pre-treatment consists of dissolving the solids in a suitable solvent, e.g. methanol treating the resulting solution with activated charcoal, or other color-removing substances, and filtering. The partially purified Antibiotic W-10 Complex is then precipitated by addition of the filtrate to a (1:1) petroleum ether:ethyl ether mixture. The resulting precipitate is chromatographed on a silica gel column using a chloroform: methanol (1:1) solvent mixture as the eluent.

The separation is monitored by sampling the column effluent, chromatographing the samples by thin layer chromatography preferably using Gelman I.T.L.C. (SA), silicic acid as the adsorbent and chloroform, methanol and water (50:50:4) as the solvent system. After detection of the antibiotics in each sample, fractions containing materials having Rf of 0.66 (0.56-0.72) are combined as are the materials having an Rf of 0.32 (0.30-0.35). On conventional silica gel plates (i.e. Analtech ^(R) TLC plates) pure samples of Antibiotic 20561 and Antibiotic 20562 have Rf values of 0.63 and 0.19, respectively, when chromatographed in the solvent system consisting of chloroform: methanol; ammonium hydroxide: water 60:30:3:2. Detection of the antibiotic is generally accomplished by exposure of the chromatogram to iodine vapors wherein the antibiotic appears as a dark spot or spots against a white background. Detection is also accomplished by placing the thin layer sheet upon an agar plate seeded with Saccharomyces cerevisiae, incubating the plate for about 16-18 hours and measuring the location of the zone or zones of inhibition. In this manner, Antibiotic 20561 and Antibiotic 20562 are separated from the substances co-produced therewith and may be obtained as crystalline solids from methanol. Antibiotics 20561 and 20562 have the physical constants and structural formulae set forth above. They assay about 800 and about 1000 meg/mg., respectively, according to the assay previously described.

Infrared

The infrared spectrum set forth in FIG. 1 is that of Antibiotic 20562 which used a Perkin-Elmer IR 221 Spectrophotometer with a sodium chloride prism and was obtained on a sample of Antibiotic 20562 in the form of a mineral oil (Nujol) mull. The characteristic absorption bands [as Wave-Numbers (W-N) in Cm⁻¹ ] of Antibiotic 20561 and of Antibiotic 20562 are as follows:

Notations; v.str.--very strong, str.--strong, m.--medium, w.--weak, v.w.--very weak, br.--broad, s.--sharp, sh.--shoulder.

                                      TABLE IV                                     __________________________________________________________________________     ANTIBIOTIC 20561     ANTIBIOTIC 20562                                          W-N        W-N       W-N         W-N                                           (Cm.sup.-1)                                                                          Notation                                                                            (Cm.sup.-1)                                                                         Notation                                                                            (Cm.sup.-1)                                                                          Notation                                                                             (Cm.sup.-1)                                                                         Notation                                 __________________________________________________________________________     3300  m., br.                                                                             1380 Nujol                                                                               3226  str., br.                                                                            1105 w.                                       2960-2860                                                                            Nujol                                                                               1255 m.   2946-2817                                                                            Nujol 1080 w.                                       1740  w.   1065 w.   1736  m., str.                                                                             1064 w.                                       1655  str.  840 w.   1667-1626                                                                            v. str., br.                                                                         1026 w.                                       1520  str.  720 w.   1546  sh.   990  sh.                                      1460  Nujol          1522  str., br.                                                                            935  v. w.                                                         1460, 1377                                                                           Nujol 889  v. w.                                                         1266-1235                                                                            m., br                                                                               837  w., v. br.                                                    1143  w.    722  w., v. br.                               __________________________________________________________________________

Nuclear Magnetic Resonance (NMR), Spectral Width-1000 Hz

Antibiotic 20562 has a characteristic NMR spectrum as shown by FIG. 2. The spectrum was obtained using a Varian XL-1 100-15 spectrometer and a Varian 620L-100 disk-based computer (Varian Associates, 611 Hansen Way, Palo Alto, California). Tetramethylsilane was used as the internal standard and the sample was dissolved in deuterated dimethylsulfoxide (DMSO d₆). For structural determination, the following absorption peaks are characteristic: (in p.p.m. from TMS), 0.87, 3H, triplet (J=7 Hz), CH₃ -CH₂ --; 1.15-0.96, 9H, overlapping doublet (J=5 Hz), CH₃ --CHOH--;1.25, 20H, --(CH₂)₁₀ --; 1.86, 6H, doublet (J=7 Hz), ##STR3## 3H, CH₃ -N<; 5.82, 2H, 2 overlapping quartets (J=7 Hz, ##STR4## doublets (J=8 Hz), ##STR5##

In a similar manner, when the NMR spectrum of Antibiotic 20561 is obtained by use of the same spectrometer and under the same conditions, the following peaks are found to be characteristic:

0.85, 3H, triplet (J=7 Hz), CH₃ --CH₂ --; 0.98-1.09, 9H, overlapping doublet (J=5 Hz), CH₃ --CH(O--)--; 1.21, 2OH, --(CH₂)₁₀ --; 1.80, 6H, doublet (J=7 Hz), CH₃ --CH═; 2.84, 3H, CH₃ --N<.

BIOLOGICAL ACTIVITY OF ANTIBIOTIC 20562

In Vitro

Anitbiotic 20562 was tested in vitro in Sabourraud dextrose broth against strains of Candida albicans, the Minimum Inhibitory Concentration (MIC) being set forth in Table IV below:

                  TABLE V                                                          ______________________________________                                         Strains of            MIC                                                      Candida albicans      (mcg/ml)                                                 ______________________________________                                         Burke                 0.075                                                    Lush                  0.075                                                    Fix                   0.075                                                    Collins               0.075                                                    Merkel                0.075                                                    29                    0.075                                                    Raub                  0.075                                                    Sperk                 0.075                                                    Bevan                 0.075                                                    Newcomb               0.075                                                    Wisconsin             0.075                                                    No. 1                 0.075                                                    ______________________________________                                    

BIOLOGICAL ACTIVITY OF ANTIBIOTIC 20561 AND ANTIBIOTIC 20562

In Vivo

The in vivo activity of Antibiotic 20561, Antibiotic 20562 and the Antibiotic W-10 complex are obtained using male CF1 mice (Carworth Famrs) given a single oral or subcutaneous does one hour after intraveneous infection with sufficient Candida albicans to kill 80% of the infected mice in 48 hours.

PD₅₀ values were determined 48 hours after infection and are as follows;

Antibiotic 20561 subcutaneous administration PD₅₀ =4 mg/kg; oral administration; PD₅₀ =25 mg/kg. Antibiotic 20562 subcutaneous administration; PD₅₀ =4 mg/kg; oral administration; PD₅₀ =25 mg/kg. Under the same test conditions Antibiotic W-10 Complex via both oral nnd subcutaneous administration had a PD₅₀ =4 mg/kg.

Toxicity

Antibiotic W-10 Complex upon oral or subcutaneous administration, exhibited an LD₅₀ greater than 50 mg/kg. Antibiotic 20561 and Antibiotic 20562 under the same test conditions exhibited an LD₅₀ of >1000 and 600 mg/kg., respectively, via subcutaneous administration and 1000 and 800 mg/kg., respectively, via oral administration.

Topical in vivo Activity of Antibiotic 20562 Against Candida Albicans Intravaginal Infection in Hamsters

Test Procedure

Using a culture of Candida albicans (Wisconsin strain), in Sabourrauds dextrose broth, infect intravaginally, five groups of six female hamsters. Forty-eight and seventy-two hours after infection, make smears of the infected animals and culture the smears. Forty-eight hours later (96 hours after infection), make additional smears, culture and commence treatment. One group of six animals (controls) receives no treatment and, therefore, remains infected throughout the test. The remaining four groups of animals are treated with Antibiotic 20562, as 1%, 5%, 10% and 15% suspensions, respectively, the vehicle for suspension being administered on a cotton swab. At the end of the three day treatment the 1%, 5% and 10% groups are 60% cured, the 15% group is completely (100%) cured.

In vivo Activity of Antibiotic 20561 and Antibiotic 20562 Against Trichophyton Mentagrophytes Infection in Guinea Pigs

The skin of fifteen guinea pigs is abraided and infected with a standard culture of Trichophyton mentagrophytes. The infected animals are then separated into three groups containing five animals each. Suspensions containing 1% of the respective antibiotics in polyethylene glycol 400 are prepared. Two of the three groups are treated topically, twice daily for ten days commencing three days after infection. The third group (controls) remained untreated throughout the test. All of the animals are tested for infection by culture and observed for skin lesions for a total of 21 days. The test results are as follows:

    ______________________________________                                                        Days to become negative                                         Compound         Culture Lesion                                                ______________________________________                                         Antibiotic 20561 4.8         6.3                                               Antibiotic 20562 5.8         9.0                                               Controls         >22         >22                                               ______________________________________                                    

The novel antibiotics of this invention, i.e. Antibiotic W-10 Complex, Antibiotic 20561 and Antibiotic 20562, are preferably administered topically, but may be administered orally or parenterally and, preferably, in admixture with a pharmaceutically acceptable carrier or excipient. The precise dose to be administered inany particular dosage form will depend upon such factors as the stage and severity of the infection, the susceptibility of the infecting organism to the antibiotic and the individual characteristics of the animal species being treated. However, in general when the compounds of this invention are administered parenterally, dosages in the range of 1 mg. to 10 mg. per kg. per day are given, usually in divided doses up to four times a day.

For topical administration, liquid suspensions, creams or ointments of 0.1% to 5% by weight are used usually as a single application each day.

For oral adminstration, the compounds of this invention are used in the form of elixirs, tablets, capsules or the like. Dosages of 1mg to 5mg per kg. are administered, usually in divided doses up to four times a day. The following are exemplary of the dosage forms in which Antibiotic W-10 Complex, Antibiotic 20561 and Antibiotic 20562 may be employed, notwithstanding the fact that for a given dosage form, only one of the above-named antibiotics is specifically designated.

    ______________________________________                                         CAPSULE                                                                        Ingredients          mg/capsule                                                                               mg/capsule                                      ______________________________________                                         Antibiotic 20562, micronized                                                                         35       175                                             Sodium Lauryl Sulfate, USP                                                                           5         25                                             Lactose, Hydrous (USP Impalpable                                                                    318       158                                             Powder)                                                                        Corn Starch (Food Grade)                                                                             40        40                                             Magnesium Stearate, USP                                                                              2         2                                                                   400 mg    400 mg                                          ______________________________________                                    

PROCEDURE

Mix the first four (4) above-named ingredients in a suitable blender for 5-10 minutes. Pass the mixture through a No. 30 mesh screen and re-blend for 5 minutes. Premix the magnesium stearate with a portion of the blended material, add the premix to the main batch and blend for 1-3 minutes. Fill the blended powder into two-piece, hard gelatin capsules using a suitable encapsulating machine.

    ______________________________________                                         TABLET                                                                         Ingredients        mg/tablet  mg/tablet                                        ______________________________________                                         Antibiotic 20562, micronized                                                                      35.0       175.0                                            Sodium Lauryl Sulfate, USP                                                                        5.0        25.0                                             Lactose, USP       307.5      147.5                                            Microcrystalline Cellulose NF                                                                     50.0       50.0                                             Corn Starch (Food Grade)                                                                          50.0       50.0                                             Polyvinylpyrrolidone                                                           (20% aqueous solution)                                                                            50.0       50.0                                             Magnesium Stearate, USP                                                                           500.0 mg   500.0 mg                                         ______________________________________                                    

PROCEDURE

Mix the first five (5) above-named ingredients in a suitable blender for 10-15 minutes. Granulate the mixture with the polyvinylpyrrolidone until a uniform damp mass is formed, (add additional water, if necessary). Pass the damp mass through a No. 8 mesh screen. Spread the damp granules on trays and dry overnight between 40°-50° C. Mill the dried granules using a suitable mill with a No. 16 mesh screen. Add the magnesium stearate to the milled granules and mix for 1-3 minutes. Compress the mixture to the specified weight (i.e., 500 mg.) on a suitable tablet press.

    ______________________________________                                         Parenteral Dosage                                                              Ingredients                  mg/ml                                             ______________________________________                                         Antibiotic 20562                     200                                       Propylene Glycol                     400                                       Glycine                              100                                       Tween 80                              5                                        Sodium Hydroxide 0.1 N                                                         or                      qs to pH 7.0                                           Hydrochloric Acid 0.1 N                                                        Water for injection     qs to 1.0 ml                                           ______________________________________                                    

PROCEDURE

Add the antibiotic as a sterile powder, to a mixture of the remaining ingredients. Adjust the pH to 7.0 using the sodium hydroxide or the hydrochloric acid, as required. Filter the formulation through a 0.22 μ membrane and aseptically fill into ampules or vials.

Alternate Procedure

Sterilize Antibiotic 20562 using ethylene oxide, or sterile filtration, or lyophilization. Transfer under aseptic conditions 200 mg of Antibiotic 20562 to each of a plurality of vials. Mix the remaining ingredients as set forth in the procedure above and sterilize by filtration through a 0.22 μ membrane. Under aseptic conditions fill into the vials containing the antibiotic.

    ______________________________________                                         Topical Composition                                                            CREAM                                                                          Ingredients          mg/g    mg/g                                              ______________________________________                                         Antibiotic 20562, micronized                                                                        10.00         50.00                                       4-Chloro-m-Cresol    2.00          2.00                                        Sodium Phosphate Monobasic                                                                          2.65          2.65                                        Monohydrate                                                                    Phosphoric Acid, NF  0.02          0.02                                        White Petrolatum, USP                                                                               150.00        110.00                                      Mineral Oil, USP (75 centistokes)                                                                   60.00         60.00                                       Polyethyleneglycol 1000 monocetyl                                                                   22.50         22.50                                       ether                                                                          Cetostearyl Alcohol  72.00         72.00                                       Water, USP qs to     1.00    g     1.00  g                                     ______________________________________                                    

PROCEDURE

Heat the mineral oil, white petrolatum cetostearyl alcohol and a portion of the polyethyleneglycol 1000 monocetyl ether to 70° l C. with agitation. In another vessel, heat about 90% of the water to 70° C. with agitation and add the 4-chloro-m-cresol, sodium phosphate and the phosphoric acid. Add the oil (mineral oil, etc.) phase to the aqueous phase at 70° C. with vigorous agitation. Cool to 60° with vigorous agitation, then to 38° C. with gentle agitation. Dissolve the remaining Polyethylene glycol 1000 monocetyl ether in water at 65° C., cool to 35° C., add Antibiotic 20562 with agitation. Add the antibiotic suspension to the cream base at 35° C. with moderate agitation and cool to room temperature (20° C.-25° C.).

    ______________________________________                                         Topical Composition                                                            OINTMENT                                                                       Ingredients           mg/g     mg/g                                            ______________________________________                                         Antibiotic 20561, micronized                                                                         10.00    10.00                                           Mineral Oil, USP (75 centistokes)                                                                    50.00    50.00                                           White Petrolatum, USP to make                                                                         1.00g    1.00g                                          ______________________________________                                    

PROCEDURE

Heat a weighed portion of white petrolatum and mineral oil to 65° C. while mixing. Cool the mixture to 50°-55° C. with stirring. Dispers the Antibiotic 20561 in the remainder of the mineral oil and mill. Add the antibiotic suspension to the petrolatum-mineral oil mixture with stirring, add the remainder of the petrolatum and cool to room temperature (20°-25° C.).

    ______________________________________                                         Solution                                                                       ______________________________________                                         Antibiotic 20562           0.2%    W/V                                         N-Methyl-2-Pyrrolidone     20.0%   W/V                                         Isopropyl-Myristate        5.0%    W/V                                         Methanol         q.s. ad   100.0%                                              ______________________________________                                    

PROCEDURE

Dissolve the antibiotic in the N-methyl-2-pyrrolidone, add the isopropyl myristate and adjust to the desired volume with methanol.

    ______________________________________                                         Gel                                                                            ______________________________________                                         Antibiotic 20562           0.2%    W/W                                         N-Methyl-2-Pyrrolidone     70.0%   W/W                                         Carbomer-934               1.0%    W/W                                         Methanol                   10.0%   W/W                                         Sodium Hydroxide 1. ON                                                                          q.s. pH   5-6                                                 Purified Water   q.s. ad   100.0%                                              ______________________________________                                    

PROCEDURE

Dissolve the antibiotic in the N-methyl-2-pyrrolidone, add methanol and water to 90% of final weight. Adjust the pH to 5-6 with the sodium hydroxide and adjust weight to 90% of final weight. Add the carbomer-934 using high speed agitation to facilitate gel formation.

    ______________________________________                                         Cream                                                                          ______________________________________                                         Antibiotic 20562             0.2%    W/W                                       N-Methyl-2-Pyrrolidone       10.0%   W/W                                       Sorbitan Monostearate        2.0%    W/W                                       Polyoxyethylene Sorbitan Monostearate                                                                       1.5%                                              Methanol                     10.0%                                             Cetopalmitate Wax            3.0%                                              Cetostearyl Alcohol          10.0%                                             2-Octyl Dodecanol            13.5%                                             Benzyl Alcohol               1.0%                                              Purified Water       q.s. ad 100.0%                                            ______________________________________                                    

PROCEDURE

Dissolve the antibiotic in the N-methyl-2-pyrrolidone and 90% of the required water. Heat the solution to 70° C. Heat the sorbitan monstearate, polyoxyethylene sorbitan monostearate, cetopalmitate wax, cetostearyl alcohol and 2-octyl dodecanol to 70° C. in a separate vessel and add the mixture to the antibiotic solution at 70° C. with rapid agitation. Cool the mixture to 45° C., then add the methanol and benzyl alcohol. Cool to 20° C. and adjust to the final weight with water.

PREPARATION OF STOCK CULTURE

Aeromonas sp. W-10

Dissolve 30 g of trypticase soy broth medium solids (Baltimore Biological Laboratories (BBL), Cockeysville, Md.) in 1.0 liter of water and sterilize. Inoculate the sterile medium with a loopful of Aeromonas sp. W-10 from an agar slant. Incubate the inoculated medium at about 35° C. for 24-48 hours with continual agitation. Freeze the culture for later use.

In a similar manner, a suitable culture may be prepared using 22 g Mueller-Hinton broth medium solids (BBL), in 1.0 liter of water, followed by sterilization, inoculation and growth.

EXAMPLE 1 Preparation of Antibiotic W-10 Complex

A. Inoculum Preparation

Dissolve 30 g of trypticase soy broth solids (BBL), in 1.0 liter of tap water and sterilize. Add 100 ml of Aeromonas sp. W-10 stock culture and incubate with agitation for 36 hours at 35° C.

B. Fermentation

Suspend the following ingredients in 10 liters of tap water in a 14 liter fermentor and sterilize: Dextrin-500 g, soybean meal, 350 g, calcium carbonate 70 g, dextrose 50 g and cobalt chloride 2.4 mg. Inoculate the medium with 500 ml of the inoculum prepared in Step A. Incubate at about 28° C. and at pH 6.5-7.0 with agitation of about 300 rpm and aeration at about 3.0 liters/minute. Commence assaying the broth after about 24 hours and continue the fermentation until peak activity is attained.

C. Isolation of Antibiotic W-10 Complex

Pool the contents of 4 ten liter fermentors which have reached peak activity and extract twice using 2 volumes (80-120 liters) of butanol saturated with water. Combine the extracts and concentrate to a residue. Filter the precipitate which usually forms during the concentration and set aside. Dissolve the residue in methanol and precipitates into a 1:1 mixture of ethyl ether-petroleum ether. Dry the precipitate to obtain thereby Antibiotic W-10 Complex in admixture with biologically inactive materials.

Yield--29 g. Assay 75 mcg/mg

EXAMPLE 2 Isolation of Antibiotic 20561 and of Antibiotic 20562

A. Partial Purification of Antibiotic W-10 Complex

Dissolve 29 g of Antibiotic W-10 Complex in 300 ml of methanol and treat with 50 g of activated carbon for one hour, (e.g. Darco G60, Atlas Powder Co., Wilmington, Del.). Filter the suspension, concentrate the filtrate and precipitate the antibiotic complex with a 1:1 petroleum ether-ethyl ether mixture. Dry the precipitate to obtain thereby Antibiotic W-10 Complex.

Yield--8.0 g Assay 200 mcg/mg

B. Chromatrography of Antibiotic W-10 Complex

Prepare a silica gel chromatography column having the dimensions 10 cm×60 cm and charge 8.0 g of Antibiotic W-10 Complex to the column. Elute the column with a chloroform-methanol 1:1 solvent mixture at the flow rate of 0.5 ml/min. Collect 20 ml fractions while monitoring the column by thin layer chromatography using a chloroform:methan:water (50:50:4) solvent system followed by bioautography. Pool the fractions having an Rf of 0.66 (0.56-0.72) and also pool the fractions having an Rf of 0.32 (0.30-0.35). Concentrate the pool resulting from the less polar fractions to a residue, dissolve the residue in methanol and precipitate with a 1:1 petroleum ether, ethyl-ether mixture. Dry the precipitate in vacuo at 40° C. to obtain thereby 415 mg of Antibiotic 20561; assay 800 mcg/mg.

Concentrate the combined more polar fractions, i.e. those having an Rf of 0.32 (0.30-0.35) and treat as described immediately above to obtain 600 mg of Antibiotic 20562; assay 800 mcg/mg.

The foregoing antibiotics, i.e. Antibiotic 20561 and Antibiotic 20562 may be further purified by crystallization from methanol.

EXAMPLE 3 Alternate Isolation of Antibiotic 20561 and Antitbiotic 20562

A. Isolation of Antibiotic W-10 Complex

Pool the contents of 6 ten liter fermentors which have reached peak activity and extract twice using 2 volumes of butanol saturated with water. Combine the extracts and concentrate until a precipitate is formed. Cool the suspension and filter. Dry the precipitate to obtain thereby Antibiotic W-10 Complex plus biologically inactive material.

Yield--29 g

B. Partial purification of Antibiotic W-10 Complex

Dissolve the product from Step A in 300 ml of methanol and treat with 50 g of activated carbon for one hour (e.g. Darco G60, Atlas Powder Co., Wilmington, Del.). Filter the suspension, concentrate the filtrate and precipitate the antibiotic complex with a 1:1 petroleum ether-ethyl ether mixture. Dry the precipitate to obtain thereby Antibiotic W-10 complex.

Yield--4.0 g

C. Isolation of Antibiotic 20561

Chromatograph the 4.0 g of Antibiotic W-10 Complex as described in Example 2, step B. Combine the fractions having an Rf of 0.66 (0.56-0.72). Concentrate to a residue, dissolve the concentrate in methanol and precipitate with a 1:1 mixture of petroleum ether and ethyl ether.

Yield--90 mg. Assay 800 mcg/mg.

M.P. 160°-166° C. [α]_(D) ²⁶ =-88° (95% pyridine, 5% H₂ O; C=0.4).

D. Isolation of Antibiotic 20562

Combine the fractions having an Rf of 0.32 (0.30-0.35), concentrate the pooled fractions to a residue and proceed as described immediately above to obtain the product of this step.

Yield--400 mg. Assay 1000 mcg/mg.

M.P. 170°-175° C. [α]_(D) ²⁶ =-60° (95% pyridine, 5% H₂ O; C=0.4). 

We claim:
 1. A process for preparing Antibiotic W-10 Complex which comprises cultivating under controlled aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen an Antibiotic W-10 Complex producing member of Aeromonas sp. W-10 until a composition of matter having substantial antifungal activity is produced and recovering the Antibiotic W-10 Complex from said medium.
 2. A process according to claim 1 wherein Aeromonas W-10 NRRL B-11053 is cultivated for from about 48 to about 90 hours.
 3. A process according to claim 2 wherein Aeromonas W-10 NRRL B-11053 is cultivated for from about 48 to about 72 hours at about 28° C., at a pH of from about 6.5-7.0 and recovering Antibiotic W-10 Complex therefrom.
 4. A process according to claim 3 including the step of separating antibiotic 20561 and Antibiotic 20562 from said Antibiotic W-10 Complex. 